Certain multiparameter biospecific assay methods have been introduced earlier. It has been common practice to use multiple labels to label biospecific reagents and to perform the separation of the signals on the basis of their different emission spectra. In most cases, however, the known multiparameter methods are based on the use of a solid support where the biospecific reagents can be immobilized at separate and optically distinguishable areas, or that are based on the use of artificial microparticles as a solid support. Some of the methods are reviewed below:
1. A method, in which various biospecific probes are attached to a matrix, which is formed by small areas on a planar solid support, is described in the patent PCT WO 84/01031. In this method, after the reaction and the wash, the signals from the photoluminescent labels in each area are measured separately, for example, using a laser scanning microscope.
2. A method, in which the identification of the analyte category is based on the color of the microparticles, which are used as a solid support and which is achieved by optically measuring the light absorption of the particle to be analyzed (J. G. Streefkerk et al. Protides Biol. Fluids 24 (1976) 811-814 and U.S. Pat. No. 5,162,863).
3. A method, in which the identification of the analyte category is performed by optically measuring the absorption of the dye inside the particle, the refractive index or the size of the particle to be analyzed (U.S. Pat. No. 5,162,863).
4. A method, in which the identification of the analyte category is based on the use of different particle sizes and in which the identification is performed by optically measuring the diameter of the particle to be analyzed (T. M. McHugh et al., Journal of Immunological Methods 95 (1986) 57-61).
5. A method, in which the microparticles are identified by means of fluorescent dyes that are mixed or impregnated within the particles, and the biospecific signal is measured from the fluorescence intensity of another fluorescent dye, such as FITC (EP 126450, GOIN 33/58).
6. A method, in which a dye emitting short decay time fluorescence (decay time a few nanoseconds), is used for the identification of microparticles, and a dye emitting long decay time fluorescence (decay time from 10 microseconds to 2 milliseconds), is used for measuring the analyte concentrations, and in which a time resolved fluorometer is used for the discrimination of the short and long life time fluorescence (U.S. Pat. No. 5,028,545).
7. A method, in which a dye emitting short decay time fluorescence (decay time a few nanoseconds), is used for the identification of the microparticles, and a molecule which generates chemiluminescence or bioluminescence (decay time several seconds), is used to measure the analyte concentrations, and in which the fluorescence and luminescent signal can effectively be separated from the fluorescence because they are excited and they emit light at different times (FI-patent 89837).
8. A method, in which a dye emitting short decay time fluorescence (decay time a few nanoseconds), is used for the identification of the microparticles and a dye emitting phosphorescence, (decay time from 10 microseconds to 2 milliseconds), is used to measure the analyte concentrations, and in which a time resolved fluorometer is used for the discrimination between the short decay time fluorescence and the long decay time phosphorescence (FI-patent 90695).
9. A method, in which dyes emitting long decay time fluorescence, such as fluorescent chelates of lanthanide ions Tb, Dy, Eu and Sm, are used for the identification of the microparticles and for measurement of the biospecific signal (FI-patent application 931198).
A common problem in many multiparameter assays mentioned above is that the signal of photoluminescent label, which indicates the analyte category, and the signal from the photoluminescent label, which measures the concentration of the biospecific probe, interfere with each other. This is a problem that significantly restricts the dynamic range of the measurement of the analyte concentration. This interference may become particularly significant when measuring low analyte concentrations and when a wide dynamic range is required for the measurement of the biospecific signal. In methods 6, 7, and 8 referred to above, interference is eliminated by choosing such photoluminescent labels for the measurement of the biospecific reaction and identification labels which have substantially different emission decay times. In methods 1, 2, 3 and 4 the analyte is identified using an alternative method rather than using a photoluminescent label. In methods 5 and 9, the identification method of the analyte essentially restricts the dynamic range of the measurement.
Another problem associated with methods 6, 7, 8 and 9 mentioned above is the long measurement time, caused by the long decay time (T1/2=1 millisecond) of the fluorescent and phosphorescent labels. This problem is caused by the saturation of the excited states of the labels, which restricts the intensity of the exciting light to such a low level that a measurement time of up to one second is needed for each microparticle. Likewise, the measurement of the signals from labels that are based on chemiluminescence, bioluminescence and electroluminescence, also take at least one second.